Alcian Blue-periodic acid-Schiff Research Articles

Spontaneous multiple cutaneous mixed tumors in Japanese giant salamander Andrias japonicus.

We examined 7 cutaneous mixed tumors in 2 wild-captured Japanese giant salamanders Andrias japonicus. The tumors were either already present and/or increased in size, or newly occurred during capativity. We sampled the 7 tumors from these animals and 3 verrucose protrusions from 3 unaffected animals, as controls, and examined them pathologically and virologically. The tumors (5 mm to 4 cm in size) were papillary protrusions or pendulated on the skin surface. The cut surface of the tumors was white, lobulated, partially hard, and contained mucus. All tumors presented similar histological characteristics of a hyaline structure and exhibited biphasic proliferation, with neoplastic epithelial cells partially composing the pseudo-ductal structure and staining positive for cytokeratin AE1/AE3. Vimentin 3B4-positive blast-like mesenchymal cells proliferated to fill the gaps in the epithelial components. Transition from unique mucous gland to neoplastic tissue was observed. The hyaline structure was stained blue by AZAN stain, Alcian blue-periodic acid-Schiff (PAS) double stain, and toluidine blue (TB) stain of pH 7.0, but was unstained by TB with pH values of 4.1 and 2.5. The mucus in the neoplastic tissue and in the mucous gland in verrucose protrusions was stained blue by Alcian blue-PAS double stain; TB staining at pH 7.0, 4.1, and 2.5 revealed metachromasy. No virus was detected in the tumors. The 7 tumors were diagnosed as cutaneous mixed tumors, and it was confirmed that the neoplastic cells originated from the mucous gland in the dermis. The biological behavior and pathological development of tumors should be elucidated because the tumors have the potential to negatively affect A. japonicus.

Changes in marine turbot (Scophthalmus maximus) epidermis and skin mucus composition during development from bilateral larvae to juvenile flat fish.

Alike other flat fish, marine turbot has the particularity that changes from larvae with bilateral symmetry to adult with asymmetry, in terms of the position of the eyes. As expected, the skin configuration of this species is also affected by the development and transformation suffered by fish during metamorphosis. In this context, changes in the epidermis of marine turbot were studied using conventional staining and histochemical techniques using six lectins (UEA-I, PNA, RCA-I, WGA, Con A and SBA). During development from larvae to juvenile (3-300 days post-hatching), the epidermis increased in both thickness and the number of cell layers. In fact, the simple cuboidal epithelium observed in larvae at day 3 already became stratified at days 10-12, which sequentially increase in thickness with fish development. Turbot epidermis is composed basically of four cell types: epithelial and mucous or secretory cells that are present through the development, and pigmented cells and a type that the authors described as club-like cells that appear during and post-metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method revealed the presence of neutral glycoconjugates in mucous and club-like cells at post-metamorphic stages of fish. Accordingly, lectin analysis showed mucous cells containing glycoproteins rich in fucose (UEA-I labelling) and glycoconjugates rich in the sequence galactose-N-acetyl galactosamine (PNA and RCA-I labelling) when this cell type appears. Interestingly, melanophores were observed in the dorsal epidermis of post-metamorphic juveniles. This type of cell contains a black-to-brown pigment that provides the skin the typical colour of this fish species. Changes in mucous coat composition were observed during fish development, which was attributed to different roles of the glycoconjugates.

COMPARISON OF HEMATOXYLIN & EOSIN (H&E) STAINING AND PERIODIC ACID SCHIFF-ALCIAN BLUE (PAS-AB) HISTOCHEMISTRY IN ESOPHAGEAL BIOPSIES IN TERMS OF INTESTINAL METAPLASIA

Barret esophagus is considered as a precursor lesion for esophageal adenocarcinomas. Histopathological examination is required for the barret esophagus, where endoscopic examination takes an important place. Intestinal metaplasia is revealed with hematoxylin & eosin (H&E) and alcian blue (AB) in histopathological examinations. There are 2 different opinions on this issue. One of them is to perform histochemical examination for AB or PAS-AB when intestinal metaplasia is suspected in routine H&E staining, while the other is to perform histochemical examination for routine AB or Periodic acid schiff alcian blue (PAS-AB) in all esophageal biopsies with H&E section. This study aims to reveal the roles of H&E and AB staining methods in the assessment of intestinal metaplasia. 200 esophageal endoscopic biopsies were included in the study. Sections of the biopsies were re-evaluated blindly by two pathologists. H&E and PAS-AB stains were compared in terms of sensitivity, specificity, positive predictivity. In statistical analysis, a strong correlation was found between H&E and AB in the evaluation of intestinal metaplasia (Kendall, p = 0.00; r = 0, 81). In H&E evaluation, sensitivity is 79%, specificity 100%, positive predictivity 100%, negative predictivity 82.6%, while sensitivity is 100%, specificity 100%, positive predictivity 100%, negative predictivity 100% in PAS-AB stained sections. The main goal in the histopathological evaluation of intestinal metaplasia is to detect positive cases. Since absence of intestinal metaplasia means less importance, higher sensitivity and negative predictivity values should be preferred rather than 100% specificity and positive predictivity values observed in H&E sections. Considering these conditions, it seems rational to use an auxiliary histochemistry containing AB.

Alcian Blue/Periodic Acid-Schiff (AB/PAS) Special Stain is a Useful Ancillary Test to Determine Neoplastic Mucin in Pancreatic Cyst Fine Needle Aspirate Biopsy Samples: A Single Institution Retrospective Study

Alcian Blue/Periodic Acid-Schiff (AB/PAS) Special Stain is a Useful Ancillary Test to Determine Neoplastic Mucin in Pancreatic Cyst Fine Needle Aspirate Biopsy Samples: A Single Institution Retrospective Study

Reduced acetylcholine and elevated muscarinic receptor 2 in duodenal mucosa contribute to the impairment of mucus secretion in 6-hydroxydopamine-induced Parkinson's disease rats.

Patients with Parkinson's disease (PD) have a higher incidence rate of duodenal ulcers. The mucus barrier provides the first line of defense for duodenal mucosal protection. However, it is unknown whether duodenal mucus secretion is affected in PD. In the present study, we used the rats microinjected 6-hydroxydopamine (6-OHDA) into the bilateral substantia nigra to investigate duodenal mucus secretion and potential therapeutic targets in duodenal ulcer in PD. Alcian blue-periodic acid-Schiff, transmission electron microscopy, immunofluorescence, duodenal mucosal incubation, and enzyme-linked immunosorbent assays were used. The 6-OHDA rats exhibited mucin accumulation and retention in duodenal goblet cells. Mucin granules were unable to fuse with the apical membranes of goblet cells, and the exocytosis ratio of goblet cells was significantly reduced. Moreover, decreased acetylcholine and increased muscarinic receptor 2 (M2R) levels were detected in the duodenal mucosa of 6-OHDA rats. Bilateral vagotomy rats were also characterized by defective duodenal mucus secretion and decreased acetylcholine with increased M2R levels in the duodenal mucosa. Application of the cholinomimetic drug carbachol or blocking M2R with methoctramine significantly promoted mucus secretion by goblet cells and increased MUC2 content in duodenal mucosa-incubated solutions from 6-OHDA and vagotomy rats. We conclude that the reduced acetylcholine and increased M2R contribute to the impaired duodenal mucus secretion of 6-OHDA rats. The study provides new insights into the mechanism of duodenal mucus secretion and potential therapeutic targets for the treatment of duodenal ulcers in PD patients.

The protective effect of NF-κB signaling pathway inhibitor PDTC on mice with chronic atrophic gastritis

Objective To understand the protective effect of NF-κB signaling pathway inhibitor pyrrolidinedithiocarbamate (PDTC) on mice with chronic atrophic gastritis (CAG). Methods Helicobacter pylori (H. pylori) infection combined with high-salt diet was used to construct the CAG mouse model, and 100 or 200 mg/kg/day PDTC was intragastrically treated for 8 weeks. Then, hematoxylin and eosin (HE) and Alcian blue-periodic acid-Schiff (AB-PAS) staining were used to observe the pathology of gastric mucosa, while immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immuno sorbent assay (ELISA) and western blotting were determined to detect the expression of related molecules. Results The nuclear content of NF-κB p65 in the gastric mucosa of the CAG mice was increased accompanying by the structural disorder of the gastric mucosal epithelium, inflammatory cell infiltration, intestinal metaplasia, and increased MUC2 expression, but the symptoms were alleviated after PDTC treatment. In addition, the expressions of TNF-α, IL-1β, IL-6 and COX2 in the gastric mucosa and serum of CAG mice were higher than those control mice, which were reduced in CAG mice treated with either 100 or 200 mg/kg PDTC. Furthermore, 100 mg/kg and 200 mg/kg PDTC treatments reduced the serum PGE2 in CAG mice with the decreased PCNA and Ki-67 expression in gastric mucosa. The therapeutic effect of 200 mg/kg PDTC was significantly better than that of 100 mg/kg PDTC. Conclusion PDTC inhibited inflammation and the excessive proliferation of gastric mucosal epithelial cells, thereby exerting a potential therapeutic effect on CAG.

Morphometrical, histological, and scanning electron microscopic investigations on the hard palate of Rahmani sheep (Ovis aries).

The present study investigated the hard palate of Rahmani sheep (Ovis aries). Samples from nine healthy adult male sheep were investigated using morphometrical, histological and scanning electron microscopic examination. Morphologically, the hard palate was elongated, narrow rostrally, and wide caudally. The incisive papilla was heart-shaped, flanked on both sides by a groove on which the nasopalatine duct opened. The palatine raphe was in the form of a groove that contained a ridge caudally. On both sides of the raphe, 13-15 pairs of palatine ridges were present and mainly occupying the narrow part. The wide part had a rough part that contains few ridges rostrally and a smooth part caudally. Histologically, the incisive papilla and palatine ridges were lined by a keratinized stratified squamous epithelium resting on a dense layer of lamina propria. The incisive papilla characterized by the presence of seromucoid salivary glands and hyaline cartilage fragments in the lamina propria. The salivary glands became abundant and well-developed in the wide part till the end of hard palate. All palatine salivary glands were Alcian blue-periodic acid-Schiff positive. By scanning electron microscopy, numerous gland openings were scattered on the surface of the palatine ridges. In conclusion, the hard palate of Rahmani sheep presented characteristic features, which may be related to the species differences, feeding behavior, and possible functional adaptations. This is the first study to report the presence of cartilaginous segments and salivary glands in the incisive papilla and provide detailed descriptions of the Rahmani sheep hard palate.

Morphology Comparison Between Goat Bone Marrow Mesenchymal Stem Cells and Adhesive Fibrin for the Repair of Annulus Fibrosus Defect of Intervertebral Discs

Introduction: The purpose of this study was to compare the histological findings of goat bone marrow mesenchymal stem cell (BMSC) transplantation and adhesive fibrin repair for annulus fibrosus defects in intervertebral discs. Material and methods: The goats were spanided into three groups: the control group, the adhesive group and the transplantation group. In the control group, surgical instruments were used to create a fibrous ring defect in the intervertebral disc of the goats. In the adhesive group, a 1.5*1.5-cm defect was also created by surgical intervention, and the broken fiber ring was then bonded with adhesive fibrin. In the transplantation group, a gelatine sponge containing the goat BMSCs was implanted into the broken annulus fibrosus, and the wound was closed layer by layer. At 6 weeks and 12 weeks after the operation, the damaged tissues were removed, and haematoxylin and eosin (HE), trichrome gelatine (Masson), Alcian blue periodic acid-Schiff (AB-PAS) and Collagen II staining was performed. Then, the tissues from the different groups were histologically compared and analyzed. Results: Goat BMSCs have a better ability to repair defects in the fibrous ring than adhesive fibrin. Over time, the number of cells or the amount of tissue following cell transplantation was greater, indicating that the degree of repair is greater with BMSCs than with adhesive fibrin. Conclusion: Histologically, repair of the defect of the fibrous ring and prevention of nucleus pulposus protrusion were more effective in the cell transplantation group than in the other two groups.

Expression of Platelet Derived Growth Factor a, Its Receptor, and Integrin Subunit Alpha V in Feline Injection-Site Sarcomas

Abstract Injection-site sarcomas are considered an important entity in veterinary oncology, especially in cats. The current study investigated the immunohistochemical expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor receptor-α (PDGFR-α), and integrin alpha subunit v in feline injection-site sarcomas (FISS). A total of 14 paraffin-embedded tissue samples previously diagnosed as soft tissue sarcomas were selected from the departmental archive and sectioned at 5µm using a microtome. Tissue sections were stained with Hematoxylin & Eosin, Alcian blue-Periodic Acid Schiff (AB-PAS), Masson’s Trichrome, and immunohistochemically for PDGFA, PDGR-α, and integrin alpha v. The majority of the tumors were fibrosarcomas (n=13/14), except one case of myxoid liposarcoma. The tumors occurred at sites commonly used for injections or vaccine administration. Histologically, tumors were characterized by peripheral infiltration of mononuclear cells mainly lymphocytes, variable sized central necrotic areas, and many multinucleated tumor giant cells. AB-PAS staining revealed the presence of myxoid or mucinous areas in (8/14) tumors while trichrome staining demonstrated a variable amount of collagenous stroma in 12/14 tumors. Vimentin immunoreactivity was observed in all the tumors while smooth muscle actin and muscle actin staining was noticed in four and two cases, respectively. PDGFA and PDGFR-α immunoexpression was observed in all 14 cases while integrin alpha v in 13/14 cases. The results of the current study indicate that fibrosarcoma is the major morphologic phenotype of FISS. The PDGFA, its receptor, and integrin alpha v immunoexpression are increased in these tumors in cats which elucidates their role in the pathogenesis of FISS.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles ameliorate airway inflammation in a rat model of chronic obstructive pulmonary disease (COPD)

BackgroundChronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin.MethodsIn this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague-Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSC-derived EVs. Lung tissue was subjected to histological analysis using haematoxylin and eosin staining, Alcian blue-periodic acid-Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student’s t test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median ± standard deviation (SD).ResultsBoth transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, and a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD.ConclusionsIn conclusion, we show that hUC-MSC-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free-based therapy for the treatment of COPD.

Rare Case of Intestinal-Type Adenocarcinoma Arising in Cervical Oesophageal Heterotopic Gastric Mucosa

Heterotopic Gastric Mucosa (HGM) also termed gastric inlet patch or inlet patch, is a rare and benign phenomenon in cervical oesophagus and can be missed during endoscopy. It has an average incidence of 2.5%. Adenocarcinoma arising in the background of gastric heterotopia is very rare and uncommon in the upper oesophagus. A 46-year-old male presented with hoarseness and progressive dysphagia for solids for the past one month. Upper Gastrointestinal Endoscopy (UGIE) revealed a tight stricture at 19-20 cm from the incisors. Initial mucosal biopsies were not conclusive. With high clinical suspicion of malignancy, patient underwent bougie dilation of oesophageal stricture followed by repeat biopsy. Histology revealed an intestinal type of adenocarcinoma, arising in a background of gastric heterotopia of the cervical oesophagus. Alcian Blue/Periodic Acid Schiff (AB/PAS) staining was positive in both the heterotopic glands and in the cancer, indicating the presence of intestinal metaplasia. Tumour cells were immunopositive for cytokeratin-7. A Positron Emission Tomography – Computed Tomography scan revealed a metabolically active lesion located in the upper third of the oesophagus along with uptake in right supraclavicular node. This case report describes a patient with primary intestinal type adenocarcinoma of the cervical oesophagus in the background of HGM not related to Barrett’s oesophagus.

Protective Effects of Korean Herbal Remedy against Airway Inflammation in an Allergic Asthma by Suppressing Eosinophil Recruitment and Infiltration in Lung.

The increasing prevalence of allergic asthma has become the world’s major health issue. Current treatments for allergic asthma focus on treating symptoms, while permanent cures still remain undiscovered. In this study, we investigated the effect of Korean traditional herbal remedy, Pyunkang-tang (PGT)—composed of six plants—on asthma alleviation in a mouse model. The PGT mixture was orally gavaged to mice (PM group, 20 mg/mouse/day) from 7 days before sensitization with ovalbumin (OVA) (day −7). On day 0 and day 14, mice from OVA-control (n = 9) and PM group (n = 8) were sensitized with OVA and alum through intraperitoneal injection. On days 18~20, OVA was challenged to mice through nasal injection and sacrificed next day. Cell profile in lung tissue was analyzed by flow cytometry and RT-qPCR analysis, and the number of eosinophils and expression of siglec-F were significantly reduced in the PM group. Lung tissue was examined with hematoxylin and eosin (H&E) and Alcian blue/periodic acid–Schiff (AB-PAS) staining. Noticeably reduced eosinophil infiltration around bronchioles was displayed in the PM group compared to the OVA-control group. Furthermore, PGT-treated mice showed a significant reduction in IL-13 and a mild reduction in IL-5 in lungs. A decreasing tendency of IL-5/13 (+) CD4+ T cells and IL-13(+) innate lymphoid cells (ILCs) and a significant reduction in IL5(+) ILCs were also observed. When treating PGT on murine lung epithelial cells stimulated by papain, there was a significant reduction in IL-33 mRNA expression levels. Taken together, oral delivery of PGT successfully alleviated asthmatic responses provoked by OVA in a mouse model and could lead to novel therapies for allergic asthma.

Qingchang Huashi Formula attenuates DSS-induced colitis in mice by restoring gut microbiota-metabolism homeostasis and goblet cell function

Ethnopharmacological relevanceInflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disease of the gastrointestinal tract, consisting of ulcerative colitis (UC) and Crohn's disease (CD). Gut microbiota and their metabolites may play a role in the pathogen of IBD, especially of the UC. Qingchang Huashi Formula (QHF), a traditional Chinese medicine formula, has shown therapeutic effect on treating UC based on the clinical practice without clear pharmacological mechanism. Aim of the studyThe aim of this study was to clearly define the effect of QHF and its components, Baitouweng (PBR) and Baizhi (ADR) on treating UC. Materials and MethodsPharmacodynamic effects of QHF and single herb were evaluated in dextran sulfate sodium (DSS) induced acute or chronic colitis mice. Body weight loss, disease activity index (DAI) and colon length were estimated. Histological changes were observed by H&E staining. The number and abundance of gut microbiota were measured with 16S rRNA sequencing. LC-MS and GC-MS were used to detect the concentration of metabolites (e.g., bile acids (BAs) and short chain fatty acids (SCFAs)). The goblet cell was observed by Alcian blue/periodic acid-Schiff (AB/PAS) straining and the crypt stem cell was estimated by immunohistochemical analyses. The colorectal tissues were used to detect levels of IL-1β, IL-6 and TNF-α by ELISA or qRT-PCR. The expression of NLRP3, Caspase 1 and IL-1β were examined by western blotting. ResultsQHF significantly inhibited colitis, protected mice from the loss of body weight and colon shorten. Comparatively, ADR and PBR showed strong efficacy in inhibiting DSS-induced colitis. We verified that while ADR was responsible for QHF's effect on maintaining gut microbiota homeostasis and metabolism, PBR was more prominent in keeping crypt stem cells proliferation and colonic goblet cells function. Moreover, we demonstrated that the alleviation of colitis by QHF was associated with the restoration of gut microbiota-metabolism homeostasis, protection of intestinal epithelial barrier and regulation of NLRP3/IL-1β pathway. ConclusionsThe finding of the present study suggested that QHF is curative in DSS-induced colitis by restoring gut microbiota-metabolism homeostasis and goblet cells function. An optimized QHF was constituted by ADR and PBR, which showed comparable efficacy on colitis to that of QHF. Our work probed out the active constitutes as well as the relevant pharmacological mechanisms of QHF, shedding light on potential new drug combination for the treatment of IBD.

Altered presence of extra cellular matrix components in murine skin cancer: Modulation by Azadirachta indica leaf extract

Background and aimAlthough, the anticancer potential of Aqueous Azadirachta indica leaf extract (AAILE) has been robustly established against cutaneous squamous cell carcinoma (SCC) in mice, however, its ability in modulating tumor associated extra cellular matrix (ECM) is largely unknown. Therefore, the present study was conceived to explore changes in ECM during murine skin cancer and its chemoprevention by AAILE. Experimental procedureSkin tumors were induced using a two-stage model of carcinogenesis employing topical application of 7,12-Dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA) as carcinogen and promoter respectively. AAILE was administered orally to the animals. Male Laca mice were divided into four groups: control, AAILE, DMBA/TPA and AAILE + DMBA/TPA. ResultsThe tumors obtained in DMBA/TPA and AAILE + DMBA/TPA groups were histologically identified as SCC. Tumor induction in these groups was accompanied by raised serum carcinoembryonic antigen (CEA) levels when compared to control counterparts. Assessment of hydroxyproline levels and histochemical staining with sirius red and trichrome stain revealed an increase in collagen in tumors of DMBA/TPA group. An increase in glycosaminoglycans (GAGs) levels was also observed in DMBA/TPA group as made evident by biochemical studies and histochemical staining using mucicarmine and alcian blue-periodic acid schiff’s stain. Administration of AAILE to DMBA/TPA treated animals caused a decrease in collagen and GAG levels along with a decrease in serum CEA levels. ConclusionSkin tumors exhibited altered presence of ECM components which is indicative of a modified ECM. AAILE administration antagonised tumor associated ECM alterations which may be contributing to its chemopreventive activity as reported previously.

Mucus characterisation in the Octopus vulgaris skin throughout its life cycle.

The development of the epidermis of octopus, Octopus vulgaris, throughout its life cycle was studied by conventional staining and histochemical techniques using lectins. The mantle, the arm and the two parts of the suckers: the infundibulum and the acetabulum were analysed independently. With the exception of the suckers, the general morphology of the epidermis does not vary from the first days post-hatching to adulthood. In general terms, histochemical techniques do not indicate changes in the composition of glycoconjugates of the epidermis main cells, epithelial and secretory cells. The epithelial cells of the mantle and arm show positivity for mannose (ConA+) in their apical portions, indicating the presence of n-glycoproteins that, among other things, provide lubrication to the surface of the body. In the suckers, the apical surface of the infundibulum contains sulphated glycosaminoglycans of the N-acetylglucosamine type that provide adhesive properties. In addition to observing three types of mucocytes, m1 and m2 are characteristic of the mantle and arm, and m3 is found in the suckers. The paralarva epidermis is characterised by the presence of Kölliker's organs whose exact function is unknown. In this study, the absence of staining with alcian blue/periodic acid-Schiff(AB/PAS) prevents the possibility of attributing a secretory function. Nevertheless, the linkage of three lectins (WGA, LEL and GSL-I) in the fascicle of the organ suggests the presence of proteoglycans rich in N-acetylglucosamine that would mainly have a structural role.

Generation of functional salivary gland tissue from human submandibular gland stem/progenitor cells

BackgroundOrgan replacement regenerative therapy based on human adult stem cells may be effective for salivary gland hypofunction. However, the generated tissues are immature because the signaling factors that induce the differentiation of human salivary gland stem cells into salivary glands are unknown.MethodsIsolated human submandibular gland stem/progenitor cells (hSMGepiS/PCs) were characterized and three-dimensionally (3D) cultured to generate organoids and further induced by fibroblast growth factor 10 (FGF10) in vitro. The induced spheres alone or in combination with embryonic day 12.5 (E12.5) mouse salivary gland mesenchyme were transplanted into the renal capsules of nude mice to assess their development in vivo. Immunofluorescence, quantitative reverse transcriptase-polymerase chain reaction, calcium release analysis, western blotting, hematoxylin–eosin staining, Alcian blue–periodic acid-Schiff staining, and Masson’s trichrome staining were performed to assess the structure and function of generated tissues in vitro and in vivo.ResultsThe isolated hSMGepiS/PCs could be long-term cultured with a stable genome. The organoids treated with FGF10 [FGF10 (+) group] exhibited higher expression of salivary gland–specific markers; showed spatial arrangement of AQP5+, K19+, and SMA+ cells; and were more sensitive to the stimulation by neurotransmitters than untreated organoids [FGF10 (−) group]. After heterotopic transplantation, the induced cell spheres combined with mouse embryonic salivary gland mesenchyme showed characteristics of mature salivary glands, including a natural morphology and saliva secretion.ConclusionFGF10 promoted the development of the hSMGepiS/PC-derived salivary gland organoids by the expression of differentiation markers, structure formation, and response to neurotransmitters in vitro. Moreover, the hSMGepiS/PCs responded to the niche in mouse embryonic mesenchyme and further differentiated into salivary gland tissues with mature characteristics. Our study provides a foundation for the regenerative therapy of salivary gland diseases.

Expression of GCRG213p, LINE-1 endonuclease variant, significantly different in gastric complete and incomplete intestinal metaplasia

BackgroundIntestinal metaplasia (IM) of the gastric mucosa is classified as complete (Type I) and incomplete IM (Type II and III) subtypes, which showed significantly different risk for developing to gastric adenocarcinoma (GAC). GCRG213, a variant of L1-endonuclease (L1-EN), first identified in our lab, was upregulated in GAC tissue. However, the relationship between GCRG213 and IM subtypes is not clear. Our study explored the association of GCRG213 protein (GCRG213p) with IM subtypes.MethodsGastric cancer and/or para-tumor tissue samples were collected from 123 patients who underwent gastrectomy for intestinal type gastric adenocarcinoma. The subtypes of IM were characterized with Alcian blue-periodic acid-Schiff and High Iron Diamine-Alcian blue staining methods. Immunohistochemistry of GCRG213p was performed, and its expression in gastric adenocarcinoma and para-tumor tissue including dysplasia, IM, and normal mucosa were analyzed.ResultsGCRG213p was expressed in 48.94% IM, 57.14% dysplasia and 55.32% GAC, respectively. GCRG213p expression was higher in well and moderately differentiated adenocarcinoma (P = 0.037). In IM glands, GCRG213p expressed mainly in the cytoplasm of absorptive enterocytes with defined brush borders, but not in goblet cells. The expression of GCRG213p in type I IM (90.00%) was significantly higher than that in type II (36.36%) and type III (25.00%) (P < 0.001). In normal gastric mucosa, GCRG213p was exclusively positive in the cytoplasm of gastric chief cells.ConclusionsThe expression of GCRG213p in complete IM was significantly higher than in incomplete IM, which implies that GCRG213p may play a role on the developing of IM to adenocarcinoma. GCRG213p was exclusively expressed in chief cells, suggesting that it might be involved in cell differentiation from the chief cells to IM.

Bifidobacterium dentium Fortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways.

Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-β, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Dendrobium Officinale Polysaccharides Protect against MNNG-Induced PLGC in Rats via Activating the NRF2 and Antioxidant Enzymes HO-1 and NQO-1.

Dendrobium officinale polysaccharides (DOP) are the main effective ingredient in Dendrobium officinale. Nuclear factor erythroid 2-related factor 2 (NRF2) signaling is regarded as an important way to mitigate the effects of reactive oxygen species (ROS) damage and inhibit gastric cancer progress. This study introduces a previously unknown effect of DOP on precancerous lesions of gastric cancer (PLGC). The mechanism discussed herein is based on the NRF2 signal pathway as well as its downstream antioxidant enzymes heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1). DOP was prepared by the alcohol deposition method, and its molecular weight was determined using High-Performance Gel-Permeation Chromatography (HPGPC). Sixty male rats were randomly divided into five groups: normal control group (NC), PLGC model group (PLGC), model treated with low dose (2.4 g/kg) of DOP (L-DOP), model treated with middle dose (4.8 g/kg) of DOP (M-DOP), and model treated with high dose (9.6 g/kg) of DOP (H-DOP). DOP was orally administered to rats for 15 consecutive days prior to the start of a seven-month course of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) exposure. Histological evaluation was observed by hematoxylin and eosin (HE) and alcian blue/periodic acid-Schiff (AB-PAS) staining. Alanine aminotransferase (ALT), aspartate transaminase (AST), serum creatinine (Scr), serum uric acid (UA), blood urea nitrogen (BUN), and HE staining were detected for liver and kidney function. The level of 8-hydroxy-deoxyguanosine (8-OHdG) in serum was detected by kits. The NRF2 protein expression was detected by immunohistochemistry, and western blotting was utilized to compare differential protein expression levels among cytoplasmic and nuclear cell fractions. Expression levels of antioxidant enzymes heme oxygenase 1 (HO-1), Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), Glutamate-Cysteine Ligase Modifier Subunit (GCLM), and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR); furthermore, the protein expression of NRF2, HO-1, and NQO-1 was detected by western blotting. The results showed that the average content of DOP is 83%, and its molecular weight is mainly contained within 3500 and 1000000. The H-DOP experimental group exhibited noticeable weight gain after seven months, reduced intestinal metaplasia, and made the atypical hyperplasia to be kept in moderate or mild degree. Data also showed DOP to be capable of decreasing levels of ALT, UA, and BUN, all of which had been elevated following the appearance of MNNG-induced PLGCs. DOP was also seen to reduce the expression of 8-OHdG and promote the expression of NRF2 in the gastric mucosa. Furthermore, RT-PCR and western blotting results showed that DOP upregulated the gene and protein expression of HO-1 and NQO-1. These findings show that DOP prevents MNNG-induced PLGC along with subsequent liver and kidney damage. The protective effects of DOP are associated with the reduction of 8-OHdG levels as well as the activation of the NRF2 pathway and its related antioxidant enzymes, HO-1 and NQO-1.